Freie Universität Berlin, Laboratory of Structural Biochemistry, Takustraβe 6, D-14195 Berlin, Germany.
Freie Universität Berlin, Laboratory of RNA Biochemistry, Takustraβe 6, D-14195 Berlin, Germany.
Nucleic Acids Res. 2020 May 7;48(8):4572-4584. doi: 10.1093/nar/gkaa182.
The single G protein of the spliceosome, Snu114, has been proposed to facilitate splicing as a molecular motor or as a regulatory G protein. However, available structures of spliceosomal complexes show Snu114 in the same GTP-bound state, and presently no Snu114 GTPase-regulatory protein is known. We determined a crystal structure of Snu114 with a Snu114-binding region of the Prp8 protein, in which Snu114 again adopts the same GTP-bound conformation seen in spliceosomes. Snu114 and the Snu114-Prp8 complex co-purified with endogenous GTP. Snu114 exhibited weak, intrinsic GTPase activity that was abolished by the Prp8 Snu114-binding region. Exchange of GTP-contacting residues in Snu114, or of Prp8 residues lining the Snu114 GTP-binding pocket, led to temperature-sensitive yeast growth and affected the same set of splicing events in vivo. Consistent with dynamic Snu114-mediated protein interactions during splicing, our results suggest that the Snu114-GTP-Prp8 module serves as a relay station during spliceosome activation and disassembly, but that GTPase activity may be dispensable for splicing.
剪接体的单一 G 蛋白 Snu114 被认为是作为分子马达或调节 G 蛋白促进剪接。然而，目前已知的剪接体复合物的结构均显示 Snu114 处于相同的 GTP 结合状态，并且目前尚不知道 Snu114 的 GTP 酶调节蛋白。我们确定了 Snu114 与 Prp8 蛋白的 Snu114 结合区域的晶体结构，其中 Snu114 再次采用了在剪接体中观察到的相同的 GTP 结合构象。Snu114 和 Snu114-Prp8 复合物与内源性 GTP 共纯化。Snu114 表现出微弱的内在 GTP 酶活性，该活性被 Prp8 Snu114 结合区域所破坏。Snu114 中的 GTP 接触残基或排列在 Snu114 GTP 结合口袋中的 Prp8 残基的交换导致酵母生长的温度敏感性，并影响体内相同的剪接事件。与剪接过程中 Snu114 介导的蛋白质相互作用的动态一致，我们的结果表明，Snu114-GTP-Prp8 模块在剪接体激活和拆卸过程中充当中继站，但 GTP 酶活性对于剪接可能不是必需的。
